Leptin is an adipocyte-derived protein that was first identified because of its central role in the regulation of body weight. Recent studies from our laboratory and others have shown that it also has important roles in regulating carbohydrate processing, including modulating insulin action and secretion. The overall goal of this proposal is to determine the mechanisms through which leptin regulates insulin secretion. To study the pathways of leptin action, we will employ LEPTIDE, a novel leptin antagonist developed and characterized in our laboratory. When given intravenously to rats, LEPTIDE causes a potent and rapid increase in insulin secretion, indicating that native leptin tonically inhibits insulin secretion. Because regulation of insulin secretion is the most rapid effect of leptin observed in vivo, its mechanism is likely to be free of the myriad secondary pathways and feedback events that can complicate analysis of leptin actions with more delayed readouts. As a result, it is an excellent system in which to explore the physiologically relevant pathways mediating leptin action. Using LEPTIDE as a tool to block leptin signaling acutely, we will explore the mechanisms of leptin's effect on insulin secretion. First, by accessing the cerebrospinal fluid in rats, we will determine whether LEPTIDE is acting through the central nervous system or in the periphery. These studies will include intracerebroventricular administration of LEPTIDE in order to isolate central nervous system effects of this protein. Second, we will determine the leptin receptor isoforms involved in the observed response to LEPTIDE, through in vivo studies on rodents with a variety of revealing leptin receptor defects, including the Koletsky rat and C57BL/KsJ db/db mice. Finally, we will explore the intracellular signaling pathways through which LEPTIDE acts, focusing on STAT-dependent RNA transcription known to be activated by leptin. Using wild-type and previously characterized mutant leptin receptors, we will examine whether differential activation of various STAT proteins accounts for the observed effects of leptin and LEPTIDE in vivo. Together, the proposed studies will help to define the pathways through which leptin regulates insulin secretion in vivo, in response to nutrient availability. Understanding these mechanisms will provide important new information about the disordered regulation of carbohydrate metabolism associated with obesity.